SUMMARY-PROJECT 3-ZUNIGA: Tissue-resident memory (Trm) CD8+ T cells are a distinct subset of memory cells that provide an essential frontline of defense against microbes at mucosal barriers and non-barrier tissues. Although Trm are now recognized to play critical roles in host defense against acute infection, their function and molecular regulation during persistent infections have been largely unexplored. This knowledge gap is fundamentally important given that infection persists primarily in non-lymphoid tissues following chronic infection. As persistent infections such as HIV, HCV and HBV are responsible for tremendous disease burden worldwide, understanding immune regulation in non-lymphoid tissues will inform novel strategies to harness Trm against chronic infections and tumors. We propose innovative approaches to elucidate new mechanisms that specifically promote Trm exhaustion, the progressive loss of effector functions, leading to persistent pathogen in tissues and enhancing susceptibility to secondary infections. These studies will likely redefine current models of CD8+ T cell differentiation, fate and function during persistent viral infection as the current understanding of T cell exhaustion is almost exclusively based on studying circulating lymphocyte populations. Specifically, we will: (1) Decipher the unique molecular regulation and heterogeneity of exhausted Trm using cutting-edge single-cell RNA-seq and small-cell-number epigenetic methodologies combined with novel integrative computational analyses. Novel regulators of exhausted Trm identified in these studies will be tested with high-throughput functional in vivo shRNA screening. (2) Elucidate the mechanisms by which T-box transcription factors, T-bet and Eomes, and the transcriptional repressor Ezh2 differentially operate to control Trm differentiation in acute and chronic infection. (3) Investigate how non-canonical TGF? signaling components regulate Trm development and exhaustion. Our studies will leverage the data and expertise represented in the other Project and Core laboratories to define the functional and molecular determinants of Trm differentiation, retention, and exhaustion, providing the foundation with which to effectively manipulate this CD8+ T cell subset to combat chronic infectious diseases and cancer where tumor infiltrating lymphocytes undergo exhaustion. As LCMV is a prototypic member of the family arenaviridae, our work will also help explain T cell suppression caused by hemorrhagic fever arenaviruses. Moreover, we anticipate that the basic knowledge on Trm regulation that will be gathered from our studies will have broad implications for other immune-related diseases in which Trm have been shown to play a pathogenic role, such as allergy and autoimmunity.